The invention relates to novel template-textured materials in the form of template-textured polymers (TTPs, (TGP)) from aqueous solutions, as well as TTPs on a solid carrier (e.g. membranes), methods for the production and use thereof for substance-specific separation of materials.
In biotechnology, for products such as enzymes, monoclonal antibodies or recombinant proteins, new and efficient separation and cleaning strategies are required. This is equally valid for synthetic drugs, in particular when these exhibit a more complex structure and/or a higher molecular weight or a restricted stability.
For all of these fields of application, substance-specific high-performance materials are searched for, a high flexibility in adapting to the specific targets being required. Solid materials (particles, films) are preferably used so as to facilitate phase separation of solid and fluid substance flows. Contrary to separation methods based on different physical properties, the chemical affinity to the carrier is the prerequisite for substance-specific separations. Substance-specificity may be obtained by biological or biomimetical receptors. For affinity separations, either specific but very sensitive biological ligands (e.g. antibodies, enzymes), or relatively unspecific synthetic ligands (e.g. dyes, metal chelates) are being used so far; examples being chromatography, solid phase extraction, membrane separation, solid phase assays or sensors (D. Sii, A. Sadana, J. Biotechnol. 19 (1991) 83).
Nonporous films, respectively layers or particles comprising affine ligands on their surfaces possess a restricted binding capacity with porous materials having a higher specific surface and binding capacity, restrictions of the binding capacity typically occur due to diffusion limitations. Directionally permeable porous membranes are therefore particularly attractive alternative materials. Established membrane methods using membranes such as micro-filtration or ultra-filtration (MF or UF) work according to the size-exclusion principle (W. Ho, K. Sirkar (Eds.), Membrane Handbook, van Nostrand Reinhold, New York, 1992). The separation of substances having a similar molecular size with porous membranes, additionally requires specific (affinity) interactions with the membrane (E. Klein, Affinity Membranes, John Wiley and Sons, New York, 1991).
The main motivation for applying affinity membranes consist in the possibility of a directional flow towards separation-specific groups (ligands/receptors), which are present in the pores in a high density. This allows for a radical improvement of efficiency (decrease in pressure, shorter turnover times, higher throughput rates, scarcely diffusion limitations in pores, faster equilibration) as compared to analogous processes using particles (D. K. Roper, E. N. Lightfoot, j. Chromatogr. A 702 (1995)3). Such affinity membranes can be used for separations of materials, preferably of proteins, but also of many other substances (e.g. peptides, nucleic acid derivates, carbohydrate or various toxins, herbicides, pesticides) and even up to cells (U.S. Pat. No. 5,766,908). Furthermore, there exist multiple application possibilities in analytics, such as, for example, for the highly selective enriching of samples or even for the decontamination of material flows (DE 19609479).
A very attractive alternative for biological or biomimetic affinity ligands/receptors, e.g. for chromatography or analytics has been developed in the past years. This is the use of specific, yet highly robust functional cavities (xe2x80x9cmolecular imprintsxe2x80x9d) in synthetic polymers produced by molecularly texturing polymerization (G. Wulff, Angew. Chem. 107 (1995) 1958; K. Mosbach, O. Ramstrxc3x6m, Bio/Technology 14 (1996) 163; A. G. Mayes, K. Mosbach, Trends Anal. Chem. 16 (1997) 321). For this purpose, a polymerization of monomers is realized in the presence of template molecules (e.g. protein, nucleic acid, low-molecular organic substance), which are able to form a relatively stable complex with a functional monomer during polymerization. After the extraction of the template, the so produced materials are able to specifically bind template molecules again. The so synthezised polymers are called template-textured polymers (TTPs, (TGP))/molecularly textured polymers (WIPs, (MIP)) or xe2x80x9cfingerprintxe2x80x9d polymers (cf. FIG. 1, FIG. 2).
In this manner, for example, the production of polymeric sorbents in the presence of smaller organic molecules (U.S. Pat. No. 5,110,833), respectively of macromolecular substances (U.S. Pat. No. 5,372,719), or the synthesis of acrylamide gels or agarose gels in the presence of proteins have been described (U.S. Pat. Nos. 5,728,296, 5,756,717). Even peptide sorbents, respectively protein-specific sorbents produced by xe2x80x9csurface-texturingxe2x80x9d of metal chelate structures on specifically functionalized particles have been described (U.S. Pat. No. 5786428). In all cases, high affinities were obtained for the respective templates. The application of artificial antibodies and receptors produced by molecular texturing, has enormous advantages, since these structures are much more stable than their natural equivalents. Moreover, they can be synthesized for each substance (even for those having less distinct antigen properties, such as small molecules or immunodepressiva), and can be produced in a considerably simpler and more cost-efficient manner than the corresponding biomolecules.
The selection of the components for the synthesis of a TTP is mainly based the interactions between template and functional monomer. Bearing the target in mind to xe2x80x9cfixxe2x80x9d these interactions as efficiently as possible and in a way accessible to affinity interactions, suitable cross-linkers and solvents are additionally selected.
Each substance having a defined three-dimensional structure (shape) may be used as a template for the synthesis of TTP. Substance classes consequently extend from small molecules having molecular weights of below or about 100 Da (e.g. herbicides) up to particles such as viruses, bacteria or cells. However, compounds having a biological function such as proteins, peptides, nucleic acids or carbohydrates are of particularly great interest. The recognition of templates by TTP is based on a combination of various factors such as reversible covalent or non-covalent binding, electrostatic and hydrophobic interactions, as well as the complementarity of the structure (shape). Which one of these factors is dominant depends on the polymeric structure, the template properties, as well as the binding conditions. In hydrophobic solvents, for example, electrostatic interactions for template recognition based on TTP are frequently dominant. In polar solvents, however, hydrophobic interactions as well as specificity of structure are most important for the template recognition. TTPs should preferably be synthesized under conditions favouring the strong, yet reversible interactions between the polymer and the template. For large molecules (100 . . . 100,000 Da), however, a combination of a plurality of weaker bonds including hydrogen bonds and hydrophobic interactions can be advantageous. For smaller molecules (50 . . . 100 Da), less strong interactions such as, for example ionic bonds, are necessary for obtaining a TTP with high affinity.
Water as solvent or aqueous systems in general are, of course, of special interest in conjunction with the above-mentioned applications. Ligand/receptor systems xe2x80x9coptimizedxe2x80x9d by nature, operate perfectly under these conditions. However, the synthesis of TTP for applications in aqueous systems, has only recently achieved an initial success (L. Andersson, Anal. Chem. 68 (1996) 111; S. Hjerten, J. L. Liao, K. Nakazato, Y. Wang, G. Zamaratskaia, H. X. Zhang, Chromatographia 44 (1997) 227). Syntheses of TTP receptors for smaller molecules cause particular problems. Up to the present moment, it is obvious that in those attempts to control not just the selection of suitable interaction agents but also the detailed arrangement of the functional groups, success has been achieved albeit only in an imperfect manner.
The invention is based on the objects to improve the known state of the art by developing template-textured materials. This task was solved in that novel template-textured polymers are synthesized, for example, on the surfaces of solid bodies as the carrier.
The present invention comprises a general polymerization method in a principally known manner, even the synthesis of TTP particles is possible under specific inventive conditions (see below). Since these particles, however, exhibit a more or less strong hydrogel character due to the preferred water-soluble monomers and the aqueous conditions (see below), the synthesis of thin TTP layers preferably is carried out on solid, preferably polymeric carriers. This surface-modification leads to covalently fixed thin layers having template imprints on its entire carrier surface, e.g. a membrane (TTM (TGM)), by means of a selectively initiated and controlled cross-linking polymerization on the carrier surface in the presence of template molecules. Due to said selectivity of the initiation, the matrix structure, respectively the pore structure, remains unobstructed. An independent optimization of the pore structure (capacity, permeability) and the surface functionality (specificity by template imprints) may therewith be achieved by means of a specific production method. Possible templates are, for example, small molecules having molecular weights of below or about 100 Da (herbicides, inter alia), larger molecules such as peptides, proteins, nucleic acids or carbohydrates, but also particles such as viruses, bacteria or cells. With filtration through or application on TTP, the templates or template-derivates may also be bound to the template imprints (functional cavities) with high specificity from diluted solutions. Thereupon, the templates or template-derivates may optionally be cleaned and subsequently either eluted under filtration conditions (as a concentrate), or may be directly identified on the carrier.
Polymeric membranes comprising a plurality of pore structures and desired mechanical etc. properties may be produced by methods such as phase inversion induced by precipitants or temperature. This allows for a selection of perfectly porous matrix membranes for the desired separation processes (E. Klein, Affinity Membranes, John Wiley and Sons, New York, 1991).
The choice of components for a template-specific TTP is preponderantly carried out on the basis of interactions between template (T) and functional monomer (FM). Bearing the target in mind to xe2x80x9cfixxe2x80x9d these interactions as efficiently as possible and in a way accessible to affinity interactions, suitable cross-linkers (C, (V)) and solvents (S, (LM)) are selected. The synthesis of the TTP layers then ensues in situ by reactive coating of the entire carrier surface, e.g. the membrane, with a reaction mixture of low viscosity, yet with maintenance of the complex consisting of T and FM. This functionalization of the membrane with TTP is carried out so as to avoid impairment of the pore structure, as well as of the stability of the matrix membrane, yet in a way that an obstruction of the (transmembrane) pores is minimized.
In order to fulfil these requirements, a photochemical initiation of a heterogenous graft copolymerization (e.g. of functional acrylates) is particularly preferred for TTP synthesis. The result is the following principal functionalization processing sequence:
1. Coating of the carrier with a photo initiator (PhI),
2. equilibration of the carrier with the reaction mixture (T, FM, C (V), S (LM), PhI), in the case of membranes: Filling and equilibrating the pore volume of the matrix membrane with the reaction mixture,
3. exposure to UV light: Selective excitation of the PhI, generation of initiator radicals on the surface of the carrier, polymerization (temperature preferably T less than 25xc2x0 C.),
4. extraction of unconverted reactands, soluble polymer and T.
These functionalizations are based on the action of the carrier material as co-initiator, i.e. that all polymers from which radicals being able to initiate a graft copolimerization may be generated by photoinitiation, can be modified in this manner. The TTP functionalizations are possible from aqueous or organic solvents. Via the initiation and polymerization conditions, functionalization degree and, hence, surface coating of the matrix can be controlled. If necessary, an obstruction of the matrix membrane pores may also be minimized in this manner in the case of TTM Thus, the application of the method arsenal established for the TTP synthesis is possible for the surface functionalization of carrier materials.
For the photofunctionalization, a hitherto unknown interaction between the adsorption of the PhI and the T on the polymeric (membrane) surface and of the polymerization was observed. From a hydrodrophilic polymer (e.g. nylon), an adsorbed hydrophobic PhI (e.g. benzophenone) can be expelled by a hydrophilic T (e.g. terbumeton); the photofunctionalization is thereby suppressed. Such a system is not appropriate for the TTP synthesis. A hydrophilic PhI (e.g. benzophenone carbon acid), on the contrary, co-adsorbs with the hydrophilic T on the hydrophilic polymer; the photofunctionalization is thereby sufficiently efficient; T binds specifically on the so synthezised TTP. A hydrophobic PhI (e.g. benzophenone) preferably adsorbs on a hydrophobic polymer (e.g. polypropylene); the photofunctionalization is efficient; for a more hydrophilic T (e.g. terbumeton), the bonds to template imprints in the graft copolymer will dominate. Various TTP structure types can be derived therefrom (cf. FIG. 3):
a) template imprints within or/and on the surface of a cross-linked graft copolymer layer, which is fixed on the carrier surface,
b)template imprints directly on the carrier surface with participation of the matrix polymer.
A chemical grafting of polymers or cross-linked polymers (e.g. synthesis of polyaniline derivates) on the surface of a matrix membrane is suitable for the production of TTP.
Templates. Suitable substance classes extend from small molecules having molecular weights of below or about 100 Da (e.g. herbicides) up to particles such as viruses, bacteria or cells. The template concentrations in the monomer mixture for TTP production are between 0.01 and 50%. By means of the present invention, ionic and electrostatic interactions, as well as hydrogen bonds can be used even in aqueous systems for the synthesis of TTP and, hence, for molecular detection. Hydrophobic interactions can produce an additional contribution. This results in significant improvements, particularly for small molecules, and can further be used for biologically relevant molecules such as, for example, amino acids, peptides, nucleic acids, oligonucleotides, sugar and oligosaccharides, and for proteins or DNA or RNA, as well.
Functional monomers having positively or negatively charged functional groups (e.g. aminoethylacrylate derivates or acrylic acid, and methacrylic acid, respectively) are suitable for TTP synthesis. In addition, hydrophobic units such as, for example, aromatic rings, cryptands or cyclodextrines may be incorporated in TTP. Complexing-capable monomers such as metal chelate complexes, Schiffs base or specific esters can also be used for the production of TTP. Aniline and derivates therefrom with further functional groups can be used for TTP synthesis. Moreover, derivates of phenyl boronic acid, for example, which are capable of forming esters with diols, are suitable as functional monomers. The concentration of functional monomers in the mixture may be between 0 and 100%. The solvents for the polymer synthesis can be the monomer itself, water (buffer), organic solvents or mixtures thereof In general, the optimum monomer type for TTP depends on the template structure and the polymerization conditions. Dependent on the conditions and the composition of the polymerization, the textured polymers can be produced in the desired density, porosity, cross-link density and consistency. Examples for cross-linkers are ethylene glycol bismethacrylate for functional acrylates, o-phenylenediamine for polyaniline, N,Nxe2x80x2-methylene bisacrylamide or piperazine bisacrylamide for acrylamide or bisepoxide for agarose. The cross-linker concentrations in the monomer mixture are between 0 and 80%.
In order to elute the template from the TTP, an acid which disturbs acid/base interactions, a saline solution having an ionic force sufficient for dissociation, or a solvent with a different polarity may, for example, be used. Thereby, the bonds complementary to the template structure are again released in the pores and/or on the surface of the polymer. However, applications of TTP having a bound template are also possible.
By selecting the strategy or conditions of functionalization, and apart from an optimum specificity of the template imprints, unspecific interactions of structurally similar or different substances with the TTP can equally be minimized. Examples therefor are the optimization of the functional monomer and/or of the cross-linker, additives in the reaction mixture (e.g. of hydrophilic monomers), multistage modifications or posterior derivations. The selectivity of the TTPs is therewith increased.
The structural characterization of the TTPs ensues in a principally known manner by means of established methods, e.g. scanning electron microscopy (SEM), FTIR-ATR-spectroscopy (Fourier Transform Infraredxe2x80x94Attenuation of Total reflexion), functional group analytics with photometric or fluorimetric methods, contact angle measurements, permeability measurements, as well as static and dynamic sorption tests with the template or other structurally similar or different substances. The static and dynamic binding capacities of the TTPs for the template, dependent on the TTP structure and the test condition (concentration, turnover time, applied substance quantities and volumes, rinsing conditions, etc.) are essential with respect to the manifold applications of the TTPs.
Templates or template derivates are bound in the template imprints with high specificity during filtration through or the application on TTP, even in high dilutions. The templates or template derivates may then, if required, be cleaned, and may subsequently be either eluted under filtration conditions (as concentrate), or may be detected directly on the carrier (cf. FIG. 4).
The following applications result from the approach according to the present invention without any limitation of the application possibilities to these specific cases:
1. Filtration (perfusion) of solutions, but also of gaseous mixtures, through TTP,
2. diffusion (dialysis) or electrodiffusion (electrodialysis) by means of TTP,
3. use of TTP in solid phase extractions, (membrane) chromatography or electrophoresis,
4. use of TTP in sensors,
5. use of TTP as catalyst,
6. application of solutions, but also of gaseous mixtures, on TTP, examples: test strips, blotting membranes, assays, drug screening.
By means of synthesized TTPs, for example, the efficient and specific reconcentration of toxic substances (herbicides) from diluted solutions is possible (cf. 1.). This can be exploited on the one hand for quantitatively eliminating such substances (detoxification); on the other hand, however, a defined enrichment (analogous to solid phase extraction; cf. 3.) for subsequent trace analysis becomes possible as well. The efficient ultra-purification of proteins, for example, a process of greatest importance in biotechnology, is likewise possible with the help of TTP (cf. 1.). Analyses, production in pure form, but also a decontamination are thereby possible as well.
The present invention allows for an improvement of the known state of the art by means of novel TTPs, which are synthesized from aqueous reaction mixtures and which also exhibit high specificities under aqueous conditions, in particular in the presence of buffer salts.
A complex between template and functional monomer, which is essentially based on ionic bonds, may easily be split by increased salt concentrations. This effect may on the one hand be used for eluting the template from the TTP, but on the other hand, restricts extremely the utilization for affinity separations, with the exception of aqueous solutions of low ionic strength.
Surprisingly, the addition of salt (e.g. buffer) during polymerization leads in this case to TTPs, having a high affinity for template molecules with a similarly high salt concentration (e.g. of the buffer under the preferred application conditions). The phenomenological aspect of this effect may be so described that the salt concentration in the reaction mixture xe2x80x9cadjustsxe2x80x9d the spacings of the functional groups participating in the ion exchange interaction. In the course of the synthesis, one obviously succeeds in fixing this advantageous constellation (synthetic receptor and ionic functional groups at a correct spacing). This method can be realized in a particularly efficient manner with surface functionalizations of solid carriers, which will be described in the following.
By selecting the strategy or conditions of functionalization, apart from an optimum specificity of the template imprints, unspecific interactions between structurally similar or different substances and the TTP, can be minimized. Examples therefor are the optimization of the functional monomer and/or of the cross-linker, additives in the reaction mixture (e.g. of hydrophilic monomers), multistage modifications or posterior derivatizations. The selectivity of the TTPs is therewith increased.
The novel template-textured materials according to the present invention, consist of template-textured polymers (TTPs) obtained by modifying the surface of solid carriers in aqueous or organic reaction solutions, and which by way of a cross-linking polymerization of functional monomers initiated on the surface of said solid carriers in the presence of a template leads to stable template imprints. These template imprints subsequently can bind template molecules or template derivatives in a specific manner, even from aqueous, salt-containing solutions.
The template-textured membranes (TTMs) are obtained by modifying the surface of membranes, which by way of a cross-linking polymerization of functional monomers initiated on the membrane surface in the presence of a template leads to stable receptor structures in the form of template imprints which subsequently are able to bind template molecules or template derivatives in a specific manner.
The inventive method for the production of template-textured materials consists in that the synthetization is carried out starting from a porous membrane with maintenance of the macroporous structure and the specific surface, and in that a high template-binding capacity, as well as a high permeability of the template-textured membrane are achieved. The synthesis of the template imprints ensues by means of a heterogenous, photoinitiated, cross-linking graft copolymerization of functional monomers on the carrier surface.
With the inventive production of template-textured materials, a substance of the H-abstraction type (abstraction of a hydrogen atom from the environment) is used as photoinitiator, and the carrier polymer is used as co-initiator, the initiation ensuing by photoexcitation of the photoinitiator.
The synthesis of the template imprints resides in a chemically initiated cross-linking polymerization of functional monomers on the carrier surface. A substance is used as initiator, which generates radicals or other initiator species for a polymerization due to chemical or physical excitation.
As carrier materials serve organic polymers such as, for example, polypropylene, polyethylene, polystyrene, polysulfone, polyamides, polyester, polycarbonate, polyacrylnitrile, polyvinylidene fluoride, polytetrafluoroethylene, polyacrylates, polyacrylamides, cellulose, amylose, agarose, as well as their respective derivatives, copolymers or blends of these polymers.
As carrier materials are used solid porous bodies such as glasses, silicates, ceramics or metals or composites thereof, even those comprising organic polymers.
The membranes preferably feature symmetrical or also asymmetrical porous structures and pore sizes between a few nm and 10 xcexcm, preferably from 100 nm up to 5 xcexcm.
As templates serve small molecules having molecular weights of below or about 100 Da, such as, for example, herbicides, active agents or amino-acids, larger molecules such as peptides, proteins, nucleic acids or carbohydrates, or even particles like viruses, bacteria or cells, and as functional monomers polymerization-capable compounds having groups enabled for interacting with templates, in particular groups comprising of carboxyl, sulfonyl, sulfate, phosphate, amino or quarternary ammonium, as well as their derivates, also in mixtures.
By a posterior or previous additional functionalization or coating, the unspecific binding of substances concurrent to template or of non-templates is decreased.
In a particular embodiment of the method according to the present invention, cross-linker monomers are also used in a mixture with the functional monomers, as well as solvents for all components of the reaction mixture.
During the production of template-textured polymers, the binding specificity and binding capacity of the template-textured polymer for the template as well as for template-similar substances may be increased by the addition of salt. As carriers films, membranes, fibres, hollow fibres, fabrics, fleece or particles are used, in each case non-porous or porous. Also a carrier-free template-textured polymer, can be produced in any optional configuration and size.
The inventive use of the novel template-textured materials resides in the separation of materials and/or the analytics of liquid or gaseous substance mixtures based on the binding of the template or template derivatives during perfusion or diffusion through template-textured polymers or the application on template-textured polymers, and further resides in the substance-specific separation of materials by means of
affinity filtration through an arrangement comprising a template-textured polymer for the concentration, purification, separation or analytical determination of substances;
dialysis or electrodialysis by means of an arrangement comprising a template-textured polymer for the concentration, purification, separation or analytical determination of substances;
solid phase extraction, chromatography, membrane chromatography or electrophoresis by means of a template-textured polymer for the concentration, purification, separation or analytical determination of substances;
a template-textured polymer as sensor or catalyst for the purification, separation or analytical determination of substances, and
a template-textured polymer as blotting membrane or test strip or material for assays or for drug screening.
The features of the present invention, apart from the claims, result also from the description, whereby the individual features in each case alone or in the form of combinations thereof represent advantageous protectable embodiments, for which protection is requested with the present specification. Said combination consists of known (membranes, polymers) and novel elements (modification of the entire surface of membranes by means of template-textured polymer layers, synthesis of thin TTP layers on solid carriers), which interact and result in an advantageous use and the aspired success in their novel overall effect, said success residing in that by means of the synthesized TTPs, for example the specific concentration of toxic substances from diluted solutions (constituting an enrichment for trace analytics), as well as for example the efficient ultra-purification of proteinsxe2x80x94a process of greatest importance for biotechnologyxe2x80x94becomes hitherto possible, and that the TTP synthesis in aqueous systems yielding a high template specificity, and the applicability of the TTP synthesis on surfaces of solid bodies as carrier can also be carried out successfully.
The invention will be described in detail by means of exemplary embodiments, without being limited to these examples.